Several recent studies indicate that loss of function (LoF) mutations in the apolipoprotein C-III (APOC3) gene offer protection from coronary heart disease (CHD) risk, possibly by reducing circulating triglyceride-rich lipoproteins (TRLs). Inhibition of the apoC-III pathway can provide an attractive therapeutic mechanism to lower CHD risk. There are however many outstanding uncertainties that exist, including impact of apoC-III deficiency on (i) lipoprotein kinetics; (ii) high density lipoprotein remodeling and functionality; (iii) non-lipid related pathways; (iv) the mechanisms by which apoC-III inhibition may reduce CHD risk, (v) the relative contribution of different lipoprotein- associated apoC-III levels on CHD; (vi) liver function and insulin resistance from a safety perspective. In this application, we propose studies to address these gaps by leveraging natural human models of apoC-III deficiency. We have identified the world?s first humans with homozygous APOC3 genetic deficiency who are from an isolated Pakistani village with a high prevalence of consanguinity. Recruitment of all inhabitants in this village (~5000 people) has already been completed; genotyping in a subset has already identified 113 APOC3 knockouts with complete apoC-III deficiency providing opportunities for detailed phenotyping. Specifically, in AIM-1, we will conduct deep phenotyping studies in 113 human APOC3 knockouts (n = 113) and in an equal number of heterozygotes and non-carriers to address the following: (i) role of apoC-III deficiency in modulating protein and lipid composition of TRLs to impact the activity of extracellular lipases; (ii) effect of apoC-III deficiency on subclasses of LDL; (iii) role of apoC-III in modulating Lp(a) levels and composition; and (iv) contribution of apoC-III deficiency to modulation of HDL composition and function. We will also address the consequences of genetic APOC3 deficiency on (iv) systemic lipid and glucose metabolism and a range of proteins related to inflammation and other pathways (v) dose-response association with atherosclerosis. In AIM-2, we will conduct lipoprotein kinetic studies using isotope tracers on 18 trios of APOC3 null homozygotes, heterozygotes, and non-carriers to evaluate the impact of apoC-III deficiency on kinetics of apoB- containing lipoproteins and production and clearance of HDL apoA-I. We will also generate human iPS cells from 5 APOC3 knockouts and 5 matched/related non-carriers and differentiate them into hepatocytes to evaluate (a) VLDL apoB and TG production (b) TRL and LDL uptake and (c) SRB1 mediated HDL uptake. In AIM-3, we will measure total plasma apoC-III and lipoprotein-associated (apoB lipoproteins, HDL, and Lp[a]) apoC-III in 5,000 participants from the EPIC-Norfolk cohort (2,500 with incident CHD events). These studies will enable assessment of: (i) disease mediation; (ii) CHD risk progressively adjusted for lipoprotein-associated apoC-III levels and other factors; (iii) dose-response association; (iv) relevance of apoC-III in subgroups (e.g., by T2D); (v) risk prediction beyond traditional CHD risk factors; and (vi) genetic loci associated with apoC-III levels.